Numerous laboratory devices have been developed to carry out filtration, chromatography and centrifugation in order to concentrate, separate and/or purify laboratory samples.
Researchers routinely need to concentrate their sample prior to other investigative research. There are two primary approaches to sample concentration: specific capture, (chromatography resin and affinity chemistries) or size exclusion filters.
For researchers using size exclusion filters, there are limitations they must accommodate in their work. The format of these devices can be either small sample volume centrifugal filters or preparative scale tangential flow separation systems. The preparation systems typically include pumps and gauges and require the user to be skilled and to monitor the process during the separation. A further limitation for the preparative systems is that the final concentrate volume can be larger (50 or more mls). Considering that the researcher's starting sample may be 250 to 1000 mls, a 50 mls concentrate is a low concentration factor. The centrifugal devices are small in scale and the sample they can conveniently process is small (less then 100 ml) because of the size limitations of the centrifuge rotor. The centrifuge devices can achieve very high concentration factors, 100× is not uncommon, but the small starting volume would require the research to monitor the separation and repeatedly refill the filter unit to concentrate the entire starting sample.
In some cases, the centrifuge device is prepared with a packed column of separation media, chromatography or affinity. These devices similarly suffer by starting volume limitations and require repeated fillings to concentrate samples greater than 100 mls.
The same limitation for process UF applies to the preparative scale chromatography media systems, in which high cost for pumps and gauges and skilled operators are required and final concentrate volumes are larger than desired.
Another low cost approach to specific capture systems is to use gravity columns. When using these systems, the researcher has to set up the system so as to achieve a head pressure sufficient to process the sample through the column. This is achieved by placing the sample to be processed in a tank and positioning it above the separation column. Tubes and connectors are used to assemble the typical gravity set up. These set ups may be inexpensive to run, but a major limitation is the processing time which can be long enough so as to require that the process be performed in a cold room to protect the sample from thermal degradation.
U.S. Pat. No. 4,755,301 discloses a centrifugal method and apparatus for concentrating macromolecules without filtering to dryness. A semi-permeable ultrafiltration membrane separates a sample reservoir from a filtrate cup, and filtrate ducts below the membrane are offset sufficiently inward from the edge of the membrane so that when the apparatus is used in a fixed angle centrifuge rotor, filtration stops once the retentate meniscus reaches the centrifugal radial level of the outermost edge of the outermost filtrate duct.
Conventional sample preparation devices are limited to relatively small sample volumes, generally about 0.5-80 milliliters.
These devices have had chromatography media added to them, typically by removing the membrane and replacing it with a more openly porous filter or frit and adding the chromatography media upstream of that filter. In some instances, a top layer of frit may be used to hold the media in the tube during storage and handling. A small sample containing a mixture of components including the desired component (generally a peptide or protein) is added to the sample reservoir and the device is then centrifuged. The desired component typically binds (is captured) to the selected media and all other material and fluid passes through the device to the filtrate reservoir. The device is removed, the filtrate is either dumped or used in further testing and a wash solution is added to the device that removes any unbound material that may have been trapped between the media or left on the surface as the fluid level dropped. This device is removed, the filtrate is dumped (generally a new filtrate cup is attached) and an elution fluid (typically a buffer at different pH or ionic strength that causes the bound desired component to release from the media) is added and spun through the device in the centrifuge. The eluent in the filtrate cup is collected as it contains the released desired component.
A problem with this type of device is that the volume that can be filtered is limited by the size of the device that can fit within the centrifuge. To filter/capture a large sample such as a liter of serum or tissue culture, one needs to carry out the above process (at least the binding step and often the washing step) multiple times.
An alternative is to use a chromatography column such as a bench scale or preparative column to process the higher volume. Such devices are expensive, and require columns, holders, pumps and sample and filtrate, wash and eluent tanks. Additionally, many of these columns are prepacked with selected media, limiting one's choices of the media available. Further, the capacity of the media used in such columns is typically much greater than what is needed for the amount of material to be captured. This means that one often wastes the extra capacity inherent in the column that is an expensive exercise. Alternatively, one can clean and store the column and use it a second time. This involves a large amount of effort to ensure that the media is fully cleaned so that no residual material (desired or contaminant) from the last run is left behind that might adversely affect the results of the next use of the column. Also most media needs to be kept refrigerated and must be kept from being contaminated during storage.
It would be desirable to provide a device and method for rapid high-quality separations or purifications of samples in a convenient and reliable manner, which can handle sample volumes considerably higher than that handled by conventional centrifugation devices and which effectively uses the correct amount of media and eliminates the need for the washing and storage of columns between runs.
It is therefore an object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample, particularly in a single pass with excellent capture.
It is another object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample and elute the component of interest at a high concentration factor, greater then 50×.
It is still another object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample, without expensive equipment and to provide a system that can reliably process the sample unattended.
It is a still further object of the present invention to provide a sample preparation device that can process relatively large volumes fast enough so as to not require refrigeration.